Sample analysis systems and methods of use thereof

ABSTRACT

The invention generally relates to sample analysis systems and methods of use thereof. In certain aspects, the invention provides a system for analyzing a sample that includes an ion generator configured to generate ions from a sample. The system additionally includes an ion separator configured to separate at or above atmospheric pressure the ions received from the ion generator without use of laminar flowing gas, and a detector that receives and detects the separated ions.

RELATED APPLICATIONS

The present application claims the benefit of and priority to each ofU.S. provisional application Ser. No. 62/074,938, filed Nov. 4, 2014 andU.S. provisional application Ser. No. 62/012,643, filed Jun. 16, 2014,the content of each of which is incorporated by reference herein in itsentirety.

GOVERNMENT SUPPORT

This invention was made with government support under DE-FG02-06ER15807awarded by the Department of Energy. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention generally relates to sample analysis systems and methodsof use thereof.

BACKGROUND

Mass spectrometry (MS) is a very sensitive analytical method and one ofthe most widely used scientific tools with applications ranging fromcomplex mixture analysis, to molecular biology and even large-scalepurification and materials preparation. A mass spectrometer works byusing magnetic and electric fields to exert forces on charged particles(ions) in a vacuum. Typically an ionization source is used to ionize ananalyte at atmospheric pressure or inside a vacuum chamber before theions are transferred to a vacuum environment of a mass spectrometerwhere the ions are focused, separated, and mass analyzed.

One challenge faced in all applications that use mass spectrometers isthe low pressure environment required for analysis. Specifically,transfer, focusing and analysis of produced ions must be done undervacuum. Accordingly, MS analysis requires expensive vacuum pumps andmanifolds to maintain a mass spectrometer under constant vacuum.Additionally, as vacuum pumps are cumbersome both physically andelectrically, this also presents a challenge in the miniaturization ofMS systems and their practical use due to the size and powerrequirements of commercially available MS platforms.

SUMMARY

The invention provides sample analysis systems that are configured toanalyze ions at or above atmospheric pressure and without the use oflaminar gas flow. Particularly, systems of the invention perform iongeneration, ion transfer/focusing, gas-phase ion/molecule reactions, ionseparation, and subsequent ion detection all in the ambient environment.

In certain aspects, the invention provides systems for analyzing asample that include an ion generator configured to generate ions from asample. The systems of the invention additionally include an ionseparator configured to separate at or above atmospheric pressure theions received from the ion generator without use of laminar flowing gas,and a detector that receives and detects the separated ions.

In certain embodiments, the ion generator includes an ionization source,and an ion injector configured to interface with the ionization sourcesuch that ions produced by the ionization source are received by the ioninjector. Typically, although not required, the ionization source isout-of-line with the detector. The ion generator may be maintained at orabove atmospheric pressure. However, in certain embodiments, the iongenerator may be maintained below atmospheric pressure. Numerousconfigurations can exist for an ion injector. In certain embodiments,the ion injector includes a cavity and one or more wire meshes thatreceive the ions produced by the ionization source.

In certain embodiments, the ion separator includes a chamber and aplurality of electrodes that are configured such that upon applicationof voltage to the electrodes, ions received from the ion injector areseparated as they travel through the chamber. In certain embodiments,the plurality of electrodes are three curved electrodes. Each of thethree curved electrodes may be separated from each other by anon-conductive spacer. In certain embodiments, at least one of the threecurved electrodes includes an opening through which a probe may beinserted.

The detector may be a mass spectrometer or a miniature massspectrometer. Alternatively, another type of ion detector may be used,such as a pixelated charge collection detector (IONCCD).

Other aspects of the invention provide methods for analyzing a sample.Those methods may involve generating ions from a sample at or aboveatmospheric pressure, separating the ions at or above atmosphericpressure without use of laminar flowing gas, and detecting the separatedions, thereby analyzing the sample. The detecting step may be at orabove atmospheric pressure.

In other embodiments, the detecting step is below atmospheric pressure.In certain embodiments, detecting includes receiving the ions into amass spectrometer or a miniature mass spectrometer.

In certain embodiments, the separating step includes transferring theions into an ion separator that includes a chamber and a plurality ofelectrodes that are configured such that upon application of voltage tothe electrodes, the ions are separated as they travel through thechamber. The plurality of electrodes may be three curved electrodes.Each of the three curved electrodes may be separated from each other bynon-conductive spacer.

Other aspects of the invention provide methods for analyzing a reactionproduct. The methods involve generating ions at or above atmosphericpressure, separating the ions at or above atmospheric pressure withoutuse of laminar flowing gas, introducing neutral molecules to theseparated ions, reacting a portion of the separated ions with theneutral molecules to produce a reaction product, and detecting thereaction product.

Another aspect of the invention provides a method for collecting ions ofan analyte of a sample that involves obtaining a sample, generating ionsof an analyte from the sample, separating the ions at or aboveatmospheric pressure without the use of laminar gas flow, and collectingthe separated ions, such as by depositing them at discrete locations ona surface.

Systems and methods of the invention can be used with any type ofsample, such as organic or non-organic, biological or non-biological,etc. In certain embodiments, the sample is derived from a biologicaltissue or is a biological fluid, such as blood, urine, saliva, or spinalcord fluid. The sample may include an analyte of interest to beanalyzed. That analyte can be native to the sample or may have beenintroduced into the sample. Exemplary analytes include therapeuticdrugs, drugs of abuse and other biomarkers. The examples herein showanalysis of therapeutic drugs, drugs of abuse and other compounds. Incertain embodiments, systems and methods of the invention can be usedfor direct analysis of biofluid samples or liquid samples.

That is, systems and methods of the invention can be used withoutperforming an sample preparation or purification steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show embodiments of a system of the invention.

FIG. 2A shows an embodiment of an ion injector. FIG. 2B shows anexemplary metal mesh.

FIG. 3 is the potential view of an elliptical geometry.

FIG. 4 panels A-B show another embodiment of an ion injector. FIG. 4panel A shows the ion injector with the ionization source and FIG. 4panel B shows the ion injector without the ionization source.

FIG. 5 panels A-C show dimensional drawings of turning electrodes (panelA), source electrode (panel B), and spacers (panel C).

FIG. 6 is a diagram illustrating how IONCCD (atmospheric pressure iondetector, commercially available from OI Analytical) data was capturedand the data assembled into a 2D plot of ion intensity from individualintegration time steps. The detector slit is indicated by the bluetransparent box.

FIG. 7 panels A-D show experimental (panels A-B) and simulated (panelsC-D) tetraalkylmmonium ion intensity at deposition surface for differentelectrode potentials. In panels A and C potentials on electrodes E₁, E₂,and E₃ were 2.90 kV, 2.60 kV, and 1.80 kV, respectively; in panels B andD potentials on electrodes E₁, E₂, and E₃ were 2.95 kV, 2.12 kV, and1.77 kV, respectively. In each case E_(source) was set to 3.00 kV andspray potential was set at 4.65 kV.

FIG. 8 is mass spectra of tetraalkyl ammonium cations from consecutiveMS scans.

FIG. 9 a graph showing simulated time of flight distribution fortetraalkylammonium cation mixture transmitted through printed electrodeassembly.

FIG. 10 panel A is additional data showing separation of tetraalkylammonium cations (C₃, C₄, C₆, C₁₂) by applying a square wave, floatedhigh voltage pulse (2950 V high, 2935 V low) to the source electrode.Spectra in FIG. 10 panels B and C are subsequent sacans from the trap ata 7.7 Hz acquisition rate.

FIG. 11 panels A-B are mass spectra showing reaction of protonatedcyclohexylamine with DMMP vapor (panel A) and tert-butylamine with DMMPvapor (panel B).

FIG. 12 panels A-B are spectra of the ion-molecule reaction in air usingelectrolytic nanoESI (Au electrode) to generate Au(ACN)²⁺ fromacetonitrile (ACN) spray solvent Neutral: 3-octyne on cotton swab (Iondetection: Thermo LTQ).

FIG. 13 illustrates simulated ion trajectories within a cylindricalelectrode with a coaxial disc electrode, each held at a potential of 1.5kV in close proximity to a surface with a potential of 20 V. Dotted lineand arrow are drawn to show axis of cylindrical symmetry.

FIG. 14 panels A-C show radial intensity of ions focused into an annulusunder varied conditions. FIG. 14 panel A is a diagram of a setup showingaxis of cylindrical symmetry and an electrode with filaments connectinga central disc electrode to an outer cylinder. FIG. 14 panel B is anexample of a 2 dimensional reconstruction of ion intensity at thedeposition surface observed when using the annular focusing electrode,which is shown in FIG. 14 panel C. FIG. 14 panel C shows the annularfocusing electrode.

FIG. 15 is a graph showing efficiency of ion transfer from ananoelectrospray ionization source to a grounded deposition surface bythe radial ion lens with different applied potentials.

FIG. 16 is a cutaway view of a 3D printed ion mobility spectrometer.

FIG. 17 panel A shows a mass spectrum of a tetraalkylammonium cationmixture transmitted through 3D printed IMS without employing separation.FIG. 17 panel B shows an ion mobility spectrum of tetraalkylammoniumcations separated using 3D printed IMS.

DETAILED DESCRIPTION

The invention generally relates to sample analysis systems and methodsof use thereof. Systems of the invention are configured to separate ionsof different mass/charge ratio in air without the use of laminar gasflow. Accordingly, systems of the invention provide a single integratedinstrument to perform ionization in air, ion transport from anionization source to a detector in air while accomplishing ion focusingand ion detection in air. In that manner, systems and methods of theinvention transfer the elements of mass spectrometry to air.

As shown herein, a system composed of small plastic electrodes wasfabricated and used to demonstrate several cases of gas-phase ionmanipulation in the open air. These manipulations highlight some of thepossible uses of 3D printed plastic electrodes for focusing and transferof ions to a mass spectrometer, including cases in which an ion/moleculereaction is performed within the electrodes at atmospheric pressure. Theseparation of ions demonstrated in the simple, low-cost systemdemonstrates that a device may be constructed in which ions may bepurified through soft-landing or directly analyzed, all without theconstraints of a vacuum system or well defined gas flow. Moreover, thedetection and two dimensional profiling of the ion beam under ambientconditions, combined with the low cost of electrode production, pavesthe way for distinct surface patterning with unorthodox electrodegeometries.

FIGS. 1A and 1B show embodiments of systems of the invention. In certainaspects, the invention provides a system 100 for analyzing a sample thatincludes an ion generator 101 configured to generate ions 105 from asample. The system additionally includes an ion separator 102 configuredto separate at or above atmospheric pressure the ions 105 received fromthe ion generator 101 without use of laminar flowing gas, and a detector104 that receives and detects the separated ions 105. In certainembodiments, the system is configured to operate in an ambient or aboveambient environment, and there is no need for a gas or gas inlet toprovide turbulent or laminar flow through the apparatus.

The ion generator 101 includes an ion injector 101 a and ionizationsource 101 b. FIGS. 1A, 1B and FIG. 4 panels A-B show exemplary ioninjectors 101 a. Each ion injector 101 a shown in these figures includesan open hollow cylindrical electrode that interfaces with ionizationsource 101 b (FIG. 4 panel A). Because the ion injector 101 a is open,it can be held at or above atmospheric pressure. In certain embodiments,within the cylindrical electrode are two metal meshes 109 (FIG. 4 panelB). The metal meshes 109 are configured such that they are separated byabout 3 mm. A floated high voltage pulse (2,530 V high, 2,480 V low) wasapplied to the mesh directly after the hollow cylindrical electrode withthe second of the two meshes held flush to an opening of the ionseparator 102 to facilitate electrical contact to the ion separator 102.Such a set-up allowed for controlled ion injection to the ion separator102. An exemplary mode operation involved a pulse width of 50 ms with arepetition rate of 1 Hz for ion injection.

Another exemplary ion injector 101 a is described for example in U.S.patent application Ser. No. 14/391,867, the content of which isincorporated by reference herein in its entirety. In these embodiments,the ion injector 101 includes an electrode having a cavity 111 (FIG.2A). There is an at least one inlet 110 within the cavity 101 aconfigured to operatively couple with an ionization source 101 b, suchthat discharge generated by the source (e.g., charged microdroplets) isinjected into the cavity 111, and is focused to an outlet. As shown inFIG. 1B, the ionization source 101 b is out-of-line with detector 104,which greatly reduces neutral transmission. The cavity 111 is shapedsuch that upon application of voltage to the electrode, ions within thecavity 111 are focused and directed to the outlet, which is positionedsuch that a proximal end of the outlet receives the focused ions and adistal end of the outlet is open to ambient pressure. The term ionincludes charged microdroplets. Generally, the outlet is grounded.

The cavity 111 can be any shape that allows for the focusing of ions. Incertain embodiments, the cavity 111 has an ellipsoidal shape. In thisembodiment, the cavity 111 is arranged such that the narrowest portionof the ellipsoid is positioned farthest from the outlet and the widestportion of the ellipsoid is positioned closest to the outlet. In otherembodiments, the cavity is a hollow half-ellipsoidal cavity, i.e., thecavity 111 is open to the air. In other embodiments, the cavity 111 isdomed shaped and connected to the outlet such that the cavity 111 sealsto the outlet. In this manner, the cavity 111 may be pressurized. Inother embodiments, the outlet is not connected to the cavity 111, ratherit is in close proximity to the opening of the elliptical cavity 111 toproduce electrical fields that facilitate the focusing of the ions inthe cavity 111 generated by the ionization source 101 b.

Ion injector 101 a may further include a gas inlet in order to produce aturbulent flow within the cavity 111. The gas flow both enhances thedesolvation of charged microdroplets to produce ions for analysis andcan assist in focusing the ions with appropriate flow fields. Ioninjector 101 a may further include a plurality of ring electrodespositioned within an interior portion of the cavity 111 such that theyare aligned with the outlet, in which the electrodes are arranged inorder of decreasing inner diameter with respect to the outlet.

The ionization source 101 b may be any ambient ionization source.Exemplary mass spectrometry techniques that utilize direct ambientionization/sampling methods including PAPERSPRAY (ion generation usingwetted porous material; Purdue Research Foundation, U.S. Pat. No.8,859,956), desorption electrospray ionization (DESI; Takats et al.,Science, 306:471-473, 2004 and U.S. Pat. No. 7,335,897); direct analysisin real time (DART; Cody et al., Anal. Chem., 77:2297-2302, 2005);Atmospheric Pressure Dielectric Barrier Discharge Ionization (DBDI;Kogelschatz, Plasma Chemistry and Plasma Processing, 23:1-46, 2003, andPCT international publication number WO 2009/102766), andelectrospray-assisted laser desoption/ionization (ELDI; Shiea et al., J.Rapid Communications in Mass Spectrometry, 19:3701-3704, 2005). Thecontent of each of these references in incorporated by reference hereinits entirety. In other embodiments, the probe operates by electrosprayionization (Fenn et al., Science 246 (4926): 64-71, 1989; and Ho et al.,Clin Biochem Rev 24 (1): 3-12, 2003) or nanoelectrospray ionization(Karas et al., Journal of Analytical Chemistry, 366(6-7):669-676, 2000).The content of each of these references in incorporated by referenceherein its entirety. In other embodiments, the probe is a paper sprayprobe (international patent application number PCT/US10/32881). In otherembodiments, the probe is a low temperature plasma probe. Such probesare described in U.S. patent application Ser. No. 12/863,801, thecontent of which is incorporated by reference herein in its entirety.

Exemplary sources include an electrospray probe or a nano-electrosprayprobe. In certain embodiments, the inlet 110 of the ion injector 101 ais configured to receive an electrospray capillary such that the spray(charged microdroplets) produced by the capillary is directly injectedinto the cavity 111 of the electrode. This is illustrated in FIG. 2A inwhich an electrospray capillary 108 is inserted within ion injector 101a and into cavity 111. In other embodiments, the ion injector 101 a isconfigured to couple with a long distance transfer line such that sprayproduced from ionization source 101 b a distance from the ion injector101 a can still be directed into the cavity 111 for focusing of ions.Long distance transfer of charged microdroplets and/or ions and devicesfor accomplishing such long distance transfer are shown for example inU.S. Pat. No. 8,410,431, the content of which is incorporated byreference herein in its entirety.

Ion injector 101 a may also include metal meshes 109 (FIGS. 2A and 2B).The metal meshes 109 are configured such that they are separated byabout 3 mm. A floated high voltage pulse (2,530 V high, 2,480 V low) wasapplied to the mesh directly after the cavity 111 with the second of thetwo meshes held flush to an opening of the ion separator 102 tofacilitate electrical contact to the ion separator 102. Such a set-upallowed for controlled ion injection to the ion separator 102. Anexemplary mode operation involved a pulse width of 50 ms with arepetition rate of 1 Hz for ion injection.

While not being limited by any particular theory or mechanism of action,an explanation of ion focusing is provided. For a given geometry, thepotential can be expressed as:

V(x,y,z) or V(r,θ,z).

Due to a cylindrical symmetry (V₉=const. for all the arbitrary x,z), thepotential can be reduced to a 2-dimensional coordinate system V(x, z).To determine whether ions are concentrated or not, two conditions mustbe matched.

$\begin{matrix}{{E_{z}(x)} = {\frac{\partial V}{\partial z} = {0{for}{all}x}}} & (i) \\\{ \begin{matrix}{\frac{\partial^{2}V}{\partial z^{2}} > {0{then}{ions}{are}{focusing}(1)}} \\{\frac{\partial^{2}V}{\partial z^{2}} = {0{then}{ions}{run}{into}{the}{focusing}{limit}(2)}} \\{\frac{\partial^{2}V}{\partial z^{2}} < {0{then}{ions}{are}{defocusing}(3)}}\end{matrix}  & ({ii})\end{matrix}$

These three cases can be easily determined by the potential graph asshown in FIG. 3. FIG. 3 is the potential view of an elliptical geometry,the circle on the left indicates case (3), the circle on the rightindicates case (1), and case (2) must be a point between the twocircles. For that analysis, it is believed that all cavity-likegeometries are able to focus ions to a certain area.

The focused ions from the ion generator 101 are transferred to the ionseparator 102. The ion separator includes curved electrodes 102 a-c,which are separated from each other by non-conductive spacers 103. Theion separator 102 operates without the use of laminar gas flow or gasflow of any type. Rather, the curvature of the electrodes produces acurved ion path and the curved electrodes are held at certain voltagesas the ions are received from the ion generator 101 and injected intothe ion separator 102. In that manner, as ions are injected into the ionseparator 102, they move along the charged curved path. The voltage actson the ions as they travel the curved path through the ion separator102, causing the ions to separate based upon their mass to charge ratio.

Each of curved electrodes 102 a-c may have a separate and distinctvoltage from its neighboring electrode and from the ion generator 101.The voltage for each of curved electrodes 102 a-c and the ion generator101 may be controlled by its own source or the voltage may be providedby one source in combination with a series of resistors to form avoltage divider. The design of the curved electrodes was based onsimulations of ion trajectories with the device, using commerciallyavailable software (e.g., SIMION 8.0; Scientific Instrument Services),which is described in the examples below. Briefly, 3D models of theelectrode assemblies were constructed and converted to stereolithography(STL) format. STL files were then converted to potential arrays usingthe SL toolbox of SIMION 8.0. Simulations were carried out using the SDSalgorithm with an arithmetic distribution of ions from 50-800 m/z (n=5).Electrodes were produced via fused deposition modeling (FDM) on a RepRapstyle 3D printer (Prusa i3) using 1.75 mm conductive ABS filament andprinted at 0.2 mm layer height. 2D images of the ion swarm at thedeposition surface were taken by scanning an IonCCD™ (OI Analytical)detector across the exit region of the electrode assembly andreconstructed based on scan rate. Ion separation was achieved byapplying a floated HV pulse on the source electrode.

The skilled artisan will appreciate that the design of the electrodesand the configuration of the system can be modified by performing othersimulations of ion trajectories using the software mentioned herein. Theskilled artisan will also recognize that the number of electrodes andspacers for the ion separator is merely exemplary, and systems of theinvention can include fewer or more curved electrodes and spacers. Thevoltage may be tuned or modified to affect the amount of ions introducedinto the ion injection region, to modify the ion beam, or to control themovement of the injected ions through the apparatus whether that be tohold or release the ions from the injection region. Electrodes may begenerated from any material that is able to hold a voltage, and is notlimited to size or shape. The dimensions of the apparatus may be furtheroptimized by modifications to the geometry and dimensions. For instance,a series of electrodes with decreasing diameters may be used toconcentrate ions to a smaller spot size at the exit electrode or thinnerand more numerous electrodes (i.e. six or more) could be used to createa more uniform electric field within the device to optimize thetransmission of ions. It should also be noted that the production ofelectrodes achieved by fused deposition modeling (FDM) allows forunconventional geometries. Additionally, with a multi-nozzle FDM printerit will be possible to construct a device consisting of multipleelectrodes and spacers that is a single solid piece. Any 3D printingdevice and available software may be useful to generate the apparatus.

The separated ions 107 are transferred from the ion separator 102 to adetector 104. Any detector that can detect ions can be used with systemsand methods of the invention. The detector may be maintained at anypressure. For example, the detector may be maintained at atmosphericpressure. In other embodiments, the detector is maintained below orabove atmospheric pressure. As shown in FIG. 1A, an exemplary iondetector 104 may be a mass spectrometer or a miniature massspectrometer. In fact, any type of mass spectrometer known in the artcan be used with systems and methods of the invention. For example, themass spectrometer can be a standard bench-top mass spectrometer. Inother embodiments, the mass spectrometer is a miniature massspectrometer. An exemplary miniature mass spectrometer is described, forexample in Gao et al. (Z. Anal. Chem. 2006, 78, 5994-6002), the contentof which is incorporated by reference herein in its entirety Incomparison with the pumping system used for lab-scale instruments withthousands watts of power, miniature mass spectrometers generally havesmaller pumping systems, such as a 18 W pumping system with only a 5L/min (0.3 m3/hr) diaphragm pump and a 11 L/s turbo pump for the systemdescribed in Gao et al. Other exemplary miniature mass spectrometers aredescribed for example in Gao et al. (Anal. Chem., 80:7198-7205, 2008),Hou et al. (Anal. Chem., 83:1857-1861, 2011), and Sokol et al. (Int. J.Mass Spectrom., 2011, 306, 187-195), the content of each of which isincorporated herein by reference in its entirety. Miniature massspectrometers are also described, for example in Xu et al. (JALA, 2010,15, 433-439); Ouyang et al. (Anal. Chem., 2009, 81, 2421-2425); Ouyanget al. (Ann. Rev. Anal. Chem., 2009, 2, 187-214); Sanders et al. (Euro.J. Mass Spectrom., 2009, 16, 11-20); Gao et al. (Anal. Chem., 2006,78(17), 5994-6002); Mulligan et al. (Chem.Com., 2006, 1709-1711); andFico et al. (Anal. Chem., 2007, 79, 8076-8082), the content of each ofwhich is incorporated herein by reference in its entirety.

In other embodiments, such as shown in FIG. 1B, the detector 104 is adetector that can detect ions at atmospheric pressure. Such an exemplarydetector is an IONCCD detector (atmospheric pressure ion detector,commercially available from OI Analytical). This exemplary detector is a2126-pixel CCD array detector that has been modified to directly detectpositively and negatively charged particles. Each pixel is 21-μm wideand 1.5-mm high with a 3-μm insulating gap. The result is a 51 mm longarray with 24-μm pitch and 88% effective area. The detector and itsoperation is further described in IonCCD 9-2013—Publication #3897, thecontent of which is incorporated by reference herein in its entirety.

In certain embodiments, the systems of the invention are configured forconducting ion/molecule reactions. To accomplish that, one or more ofelectrodes 102 a-c have an opening through which a probe may be inserted(FIG. 1A and FIG. 4 panel A). For example, FIG. 1A shows a configurationin which one of the electrodes of the ion separator 102 includes anopening 105 through which probe 106 can be inserted. Probe 106 includesone or more reactants that will interact with ions 107 as those ionspass through ion separator 102. A reaction product is produced that isdetector by detector 104. The skilled artisan will appreciate that thesystem configuration shown in FIG. 1A is exemplary. In certainembodiments, more than open opening is used, for example, more than oneof electrodes 102 a-c include an opening. The opening can also be on anyportion of electrodes 102 a-c and can be located on any one ofelectrodes 102 a-c. Additionally, the opening can be any size, and thesize will typically be based on the size of the probe that needs to beinserted into the opening.

Systems and methods of the invention can be used with any type ofsample, such as organic or non-organic, biological or non-biological,etc. In certain embodiments, the sample is derived from a biologicaltissue or is a biological fluid, such as blood, urine, saliva, or spinalcord fluid. The sample may include an analyte of interest to beanalyzed. That analyte can be native to the sample or may have beenintroduced into the sample. Exemplary analytes include therapeuticdrugs, drugs of abuse and other biomarkers. In certain embodiments,systems and methods of the invention can be used for direct analysis ofbiofluid samples or liquid samples. That is, systems and methods of theinvention can be used without performing any sample preparation orpurification steps.

Systems and methods of the invention are also useful for producing andseparating ions in air that can be collected (soft landed) on surfacesfor use as reagents for chemical reactions occurring at surfaces.Systems and methods for collecting ions are shown in Cooks, (U.S. Pat.No. 7,361,311), the content of which is incorporated by reference hereinin its entirety. In particular embodiments, systems and methods of theinvention are coupled with nanoESI probes because nanoESI probes use alow flow rate such that molecular ions of low internal energy areproduced, thus avoiding fragmentation.

Systems and methods of the invention allow for the capture of intactpolyatomic ions at a condensed phase interface—and reactive ion/surfacecollisions. The surfaces can subsequently be analyzed. Surfacecharacterization methods include keV energy ion sputtering (SIMS),temperature programmed desorption (TPD), and surface enhanced Ramanspectroscopy (SERS). Systems and methods of the invention can be used toinvestigate any chemical system. Exemplary chemical systems that can beinvestigated using apparatuses of the invention include olefinepoxidation, transacylation, aza-Diels-Alder reactions and nitrogenfixation into alkanes.

Another use for the invention is for altering chemical functionalitiesat a surface. Ions and charged droplets impinging on a surface have beenshown to increase the efficiency and rate of chemical reactionsoccurring at the surface (Abraham et al., Journal of the AmericanSociety of Mass Spectrometry, 2012, 23, 1077-1084; Abraham et al.,Journal of the American Society of Mass Spectrometry, 2012, 23, 842-849;and Abraham et al., Angewandte Chemie International Edition, 2012, 51,1-6). This, when coupled with ion separating with apparatuses andmethods of the invention at or above atmospheric pressure, allows forembodiments in which ions are used to alter the chemical functionalitiesat a surface in a spatially resolved manner, all performed atatmospheric pressure. One example of such a case is the site-specificsilylation of a glass surface via reactions of silylation agents (incharged droplets, or as free ions) with hydroxyl groups present on theglass to create hydrophobic areas. When combined with ambient ionseparating, spatially controlled chemically specific surfacemodification can be achieved at atmospheric pressure. This capability isnot limited to silylation chemistry, which serves simply as one exampleof the chemistry possible.

Collection of Ions

Systems and methods for collecting ions are shown in Cooks, (U.S. Pat.No. 7,361,311), the content of which is incorporated by reference hereinin its entirety. Generally, the preparation of microchips arrays ofmolecules first involves the ionization of analyte molecules in thesample (solid or liquid). The molecules can be ionized by any method.The ions can then be separated and collected using systems and methodsdescribed herein.

To achieve this, a microchip or substrate is moved or scanned in the x-ydirections and stopped at each spot location for a predetermined time topermit the deposit of a sufficient number of molecules to form a spothaving a predetermined density. Alternatively, the gas phase ions can bedirected electronically or magnetically to different spots on thesurface of a stationary chip or substrate. The molecules are preferablydeposited on the surface with preservation of their structure, that is,they are soft-landed. Two facts make it likely that dissociation ordenaturation on landing can be avoided. Suitable surfaces forsoft-landing are chemically inert surfaces that can efficiently removevibrational energy during landing, but which will allow spectroscopicidentification. Surfaces which promote neutralization, rehydration orhaving other special characteristics might also be used for proteinsoft-landing.

Generally, the surface for ion landing is located after the ionseparator or alternatively after the detector. Without a detector,separated ions are landed directly onto a surface. When using a massspectrometer as a detector, the high voltages on the conversion dynodeand the multiplier are turned on and the ions are detected to allow theoverall spectral qualities, signal-to-noise ratio and mass resolutionover the full mass range to be examined. In the ion-landing mode, thevoltages on the conversion dynode and the multiplier are turned off andthe ions are allowed to pass through the hole in the detection assemblyto reach the landing surface of the plate (such as a gold plate). Thesurface is grounded and the potential difference between the source andthe surface is 0 volts.

An exemplary substrate for soft landing is a gold substrate (20 mm×50mm, International Wafer Service). This substrate may consist of a Siwafer with 5 nm chromium adhesion layer and 200 nm of polycrystallinevapor deposited gold. Before it is used for ion landing, the substrateis cleaned with a mixture of H₂SO₄ and H₂O₂ in a ratio of 2:1, washedthoroughly with deionized water and absolute ethanol, and then dried at150° C. A Teflon mask, 24 mmx 71 mm with a hole of 8 mm diameter in thecenter, is used to cover the gold surface so that only a circular areawith a diameter of 8 mm on the gold surface is exposed to the ion beamfor ion soft-landing of each mass-selected ion beam. The Teflon mask isalso cleaned with 1:1 MeOH:H₂O (v/v) and dried at elevated temperaturebefore use. The surface and the mask are fixed on a holder and theexposed surface area is aligned with the center of the ion optical axis.

Any period of time may be used for landing of the ions. Between eachion-landing, the Teflon mask is moved to expose a fresh surface area,and the surface holder is relocated to align the target area with theion optical axis. After soft-landing, the Teflon mask is removed fromthe surface.

In another embodiment a linear ion trap can be used as a component of asoft-landing instrument. Ions travel through a heated capillary into asecond chamber via ion guides in chambers of increasing vacuum. The ionsare captured in the linear ion trap by applying suitable voltages to theelectrodes and RF and DC voltages to the segments of the ion trap rods.The stored ions can be radially ejected for detection. Alternatively,the ion trap can be operated to eject the ions of selected mass throughthe ion guide, through a plate onto the microarray plate. The plate canbe inserted through a mechanical gate valve system without venting theentire instrument.

The advantages of the linear quadrupole ion trap over a standard Paulion trap include increased ion storage capacity and the ability to ejections both axially and radially. Linear ion traps give unit resolution toat least 2000 Thomspon (Th) and have capabilities to isolate ions of asingle mass/charge ratio and then perform subsequent excitation anddissociation in order to record a product ion MS/MS spectrum. Massanalysis will be performed using resonant waveform methods. The massrange of the linear trap (2000 Th or 4000 Th but adjustable to 20,000Th) will allow mass analysis and soft-landing of most molecules ofinterest. In the soft-landing instrument described above the ions areintroduced axially into the mass filter rods or ion trap rods. The ionscan also be radially introduced into the linear ion trap.

The ions can be separated in time so that the ions arrive and land onthe surface at different times. While this is being done the substrateis being moved to allow the separated ions to be deposited at differentpositions. The ions can be directed to different spots on a fixedsurface by scanning electric or magnetic fields.

It is desirable that the structure of the analyte be maintained duringthe soft-landing process. On such strategy for maintaining the structureof the analyte upon deposition involves keeping the deposition energylow to avoid dissociation or transformation of the ions when they land.This needs to be done while at the same time minimizing the spot size.Another strategy is to mass select and soft-land an incompletelydesolvated form of the ionized molecule. Extensive hydration is notnecessary for molecules to keep their solution-phase properties ingas-phase. Hydrated molecular ions can be formed by electrospray andseparated while still “wet” for soft-landing. The substrate surface canbe a “wet” surface for soft-landing, this would include a surface withas little as one monolayer of water. Another strategy is to hydrate themolecule immediately after mass-separation and prior to soft-landing.One strategy is to deprotonate the mass-selected ions using ion/moleculeor ion/ion reactions after separation but before soft-landing, to avoidundesired ion/surface reactions or protonate at a sacrificialderivatizing group which is subsequently lost.

Different surfaces are likely to be more or less well suited tosuccessful soft-landing. For example, chemically inert surfaces whichcan efficiently remove vibrational energy during landing may besuitable. The properties of the surfaces will also determine what typesof in situ spectroscopic identification are possible. The ions can besoft-landed directly onto substrates suitable for MALDI. Similarly,soft-landing onto SERS-active surfaces is possible.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

The Examples herein illustrate systems and methods in which ions aregenerated and manipulated in the ambient environment using polymericelectrodes produced with a consumer-grade 3D printer. The ability tofocus, separate, react, and detect ions in the ambient environment isdemonstrated and the data agree well with simulated ion behavior. Thiswork demonstrates ion generation, ion transfer/focusing, gas-phaseion/molecule reactions, ion separation, and subsequent ion detection allin the ambient environment using plastic electrodes produced via rapidprototyping. In some Examples, the device is used to prepare ions formass analysis in a mass spectrometer while in other cases it is used ina stand-alone fashion as a reactor/analysis system.

Example 1: Electrodes and Materials

Electrodes were printed at 200 μm layer height with an FDM 3D printer(Prusa i3v, Makerfarm) from 1.75 mm conductive ABS filament(Makergeeks). Machine code (g-code) generation for the printed partproduction was performed in Slic3r v1.1.6 with a 25% hexagonal infill.All nanoESI emitters were pulled from 1.5 mm OD, 1.1 mm ID borisilicateglass capillaries on a Sutter P-97 micropipette puller to a final tipdiameter of 5 μm. Dimethyl methylphosphonate, tetradodecylammoniumbromide, and tetrahexylammonium bromide were purchased from FlukaAnalytical. Cyclohexylamine, and tetrabutylammonium bromide acquiredfrom Eastman Chemical. HPLC grade acetonitrile and methanol werepurchased from Sigma-Aldrich and Macron Fine Chemicals, respectively.

Example 2: Fundamentals of Ion Motion at Atmospheric Pressure andSimulation Environment

The simulation of gaseous ion trajectories has been used extensively inthe development of ion optics for MS, IMS, electron microscopes (EM),and focused ion beam (FIB) systems. In the case of systems operating inhigh-vacuum (EM and FIB) the simulation environment is often simplifiedand assumed to be collision-free and ion motion is influenced purely byelectric and magnetic fields; however, IMS and many MS systems operatein a pressure regime in which collisions cannot be neglected. SIMION 8.0includes two collision models (HS1 and SDS) that can be incorporated forthe treatment of these collisions. HS1 employs hard-sphere collisionkinetics to compute the resulting ion trajectory change for ion-moleculecollisions individually. This approach is not computationally feasibleat atmospheric pressures as the mean free path in air at 25° C. isapproximately 67 nm². Rather than treating individual collisions, theSDS algorithm uses a combined approach of diffusion and ion mobility tosimulate ion motion in electric fields.

The motion of ions at atmospheric pressure is heavily influenced by thediffusion of ions in the medium, as well as by external forces exertedon the ions (electric fields, bulk gas flow, etc.). Diffusion can beexpressed as:

J=D∇ _(n)  Eq. 1

where J, D, and ∇_(n) are the number of ions passing through an areanormal to the gas flow, a proportionality constant, and theconcentration gradient, respectively. In the SIMION-SDS algorithm,diffusion is simulated by imposing a random ion jump onto the iontrajectory. The radius of the jump is determined by an interpolationbetween collision statistics tables (selected based on the mass ratio ofthe ion to a background gas molecule) and scaled appropriately based onan expected number of collisions in the simulation time step.

When subjected to an electric field (E), the velocity of an ion in a gaswith no bulk flow is determined by its mobility (K) in the buffer gas.

ν=KE  Eq. 2

K is determined experimentally and is directly proportional to D and thecharge (e) on the ion and inversely proportional to temperature (T)multiplied by the Boltzmann constant (k)³.

$\begin{matrix}{K = \frac{eD}{kT}} & {{Eq}.3}\end{matrix}$

This is known as the Nernst-Townsend relation and holds for the cases inwhich ions are thermalized. The mobility can further be expressed as:

$\begin{matrix}{K = {\frac{3e}{16N}( \frac{2\pi}{\mu{kT}} )^{1/2}( \frac{1}{Q_{D}} )}} & {{Eq}.4}\end{matrix}$

Where N is the density of the neutral molecules, μ is the reduced massof the collision pair, and Q_(D) is the collisional cross section. Dueto the range of working conditions used in IMS instruments, the mobilityof an ion is often reported as the reduced mobility (K₀) which iscorrected for 273 K and a pressure (P) of 760 Torr:

$\begin{matrix}{K_{0} = {{K( \frac{273}{T} )}( \frac{P}{760} )}} & {{Eq}.5}\end{matrix}$

At each time step within a SIMION-SDS simulation, the velocity of an ionis subjected to the effects of gas flow and the applied electric field,in the form of mobility (Eq. 2). A simulated diffusion in the form of arandom jump is superimposed on this motion to determine the location ofthe ion during the start of the next time-step. A more detaileddiscussion of the SDS algorithm is provided in the literature and in theSIMION 8.0 documentation. The SDS algorithm is capable of either using adefined mobility for each ion, or in the cases in which this data is notavailable, known information (particle diameters, masses, etc.) is usedto estimate a value for ion mobility. Spatial variations in gas flow,pressure, and temperature may also be incorporated into the SDSalgorithm to more accurately model conditions in which these parametersare known. Effects due to space charge can also be included in themodeling; however, ions must be flown as a group when incorporatingspace charge effects into an SDS simulation. For all simulationsperformed in this work, bulk gas flow was assumed to be zero and spacecharge was not considered in order to decrease computational time.

Example 3: Simulation of 2D Ion Distribution at Deposition Surface

The potential arrays (PA) used in the simulation of both the 2D iondistribution at the deposition surface (described below) and theseparation of tetraalkylammonium (TAA) cations (described below) weregenerated from .stl fomat using the “Convert STL->PA” option in theSIMION 8.0 SL Tools. A 0.2 mm/grid unit resolution was selected to mimicthe 0.2 mm layer height resolution at which the electrodes were printedand the solid strategy was set as “solid points under surface normal”.

For the simulation of the mesh used for ion injection in the TAA cationseparation, the mesh was modeled as a plane with a thickness equal tothe grid cell size (0.2 mm). Because SIMION treats a plane of 1 gridunit as a 100% transmission ideal grid, this means that the simulatedmesh passes ions at all points. This simplification was made to decreasecomputational time in the simulation as a smaller unit cell size (<0.2mm/grid unit) is required to model the woven mesh in its true form whichwould result in a much larger PA space.

In the case of the simulated 2D ion distribution at the depositionsurface, ions were initiated with a 3D Gaussian distribution (σ_(x),σ_(y), and σ_(z)=5 mm) in the center of the source electrode with asingle time of birth (TOB). The contour plots of simulated iondistribution (FIGS. 2c and 2d ) were generated using a histogram binwidth of 0.05 mm×0.05 mm.

The simulation of ion separation was performed by initiating all ionswith a uniform cylindrical distribution within the area between the twomesh electrodes. At the start of the simulation time step, the injectionvoltage was set to the high value used for injection (2530 V) for 50 ms,after which it was set to the low value (2480 V) for the remainder ofthe simulation time period. This approach is not meant to accuratelymodel the ion distribution between the mesh electrodes during theinjection, but is used to simplify the simulation so that arrival timesof ions at the detector (a mass spectrometer in this case) can beapproximated to determine if simulation values agree with experimentaldata.

Example 4: Electrode and Spacer Dimensions

FIG. 5 panels A-C show dimensional drawings of turning electrodes (panelA), source electrode (panel B), and spacers (panel C).

Example 5: Ion Injector

Injection of ions into the turning electrodes was accomplished bymodifying the electrode system to include a region separated by two wiremeshes (FIG. 4 panel B). The mesh closest to the source electrode washeld at a low value of 2480 V which prevented the transmission of ionsinto the curved electrodes. Ions were injected into the curved electroderegion by pulsing this voltage to a value of 2530 V for 50 ms.

Example 6: Profiling of Beam Using IONCCD (Atmospheric Pressure IonDetector, Commercially Available from OI Analytical)

The ion beam exiting the electrode assembly was profiled by mounting theIONCCD (atmospheric pressure ion detector, commercially available fromOI Analytical) detector on a moving stage and scanning the detector slitacross the exit of the last electrode at a fixed rate of 0.100 mm/s. Adiagram of this process is shown in FIG. 6. A potential of 10 V wasapplied to the detector housing during the signal acquisition. Thispotential served to increase the electric field strength between thefloated detector array and the aluminum enclosure (0.711 mm gap betweenhousing and detector array), thus drawing ions to the detector surfacewhich results in a stronger signal.

The 2D intensity plots were reconstructed from the data by a Matlabscript which uses the integration time (100 ms in all experiments) alongwith the velocity of the moving stage to calculate position along thescan axis (Y(t)) for each detection cycle of the detector.

The elongation seen in the reconstructed intensity plots (FIG. 7 panelsA-D) when compared to the simulated 2D ion distribution at the electrodeexit is likely a result of both the gap between the stainless steelhousing and the detector surface as well as the width of the pixel array(1 mm). The elongation in the pixel axis is likely the result of ionsdiffusing outward after entering the gap between the housing and thedetector surface. The 10 V potential serves to negate this effectslightly, but application of larger potentials poses the risk ofdamaging the detector electronics. The width of the pixels mostcertainly contributes to an elongation in the reconstructed signalintensity along the scan axis. For example, at a scan rate of 0.100 mm/sthe detector slit moves approximately 0.100 mm during an integrationtime step which is only 10% of the pixel width. This means that theposition assigned to each integration time step along the scan axis alsoincludes the entirety of ions exiting the electrode ±500 μm from theassigned position (Y(t)).

Example 7: System Configuration

A curved electrode system was constructed from a conductive polymerusing a fused deposition modelling (FDM) 3D printer. The assemblyconsists of a cylindrical source electrode region (E_(source)) with aninner diameter (ID) of 20 mm and a length of 30 mm, proceeded by 3curved electrodes (E_(n)) with an ID of 15 mm and a swept angle of 45degrees around a 15 mm radius of curvature. All electrodes are separatedby 3 mm with spacers printed in either acrylonitrile butadiene styrene(ABS) or polylactic acid (PLA). Dimensional drawings are provided inFIG. 5. The electrode assembly (FIG. 1A) serves to focus ions from aspray source to a well-defined region with the application of anappropriate potential gradient along the ion path. The device is shownin FIG. 1A interfaced with the inlet of a mass spectrometer along with acutaway rendering with an overlay of simulated ion trajectories (FIG.1B). The curvature of the ion path greatly reduces the probability ofneutral transmission by avoiding line-of-sight from the sprayer to thedetection/deposition surface. Although this geometry is difficult tomachine using traditional subtractive manufacturing techniques, it istrivial to produce by additive manufacturing methods such as FDM. FDM isa process by which a plastic filament is extruded through a heatednozzle to form an object one layer at a time (Gibson et al., SpringerUS, 2010, pp. 160-186). Common materials used in FDM include ABS, PLA,polyamide (nylon), polyethylene terephthalate (PET), polycarbonate (PC),etc. FDM was chosen for the construction of the electrode assemblybecause of its low cost, the availability of a range of materials, andthe rapid nature in which the parts could be produced; the entireassembly could be constructed in under 3 hours.

Simulations of ion trajectories within the device were performed withSIMION 8.0 (Scientific Instrument Services) and the included statisticaldiffusion simulation (SDS) algorithm (further described in Example 1above). At atmospheric pressure, it is not computationally efficient toutilize traditional hard-sphere collision models as the mean free pathin air is ˜67 nm. Instead, the SDS algorithm calculates ion motion basedon ion mobility and a simulated diffusion in the form of “jumps” in arandom direction at each time step, the magnitude of which is determinedbased on collision statistics. A more detailed discussion of iontrajectory simulation at atmospheric pressure and details of simulationparameters used are included in Example 1. The workflow from design tosimulation involved the use of the SL toolkit included with SIMION. TheSL toolkit allows the user to import geometry in the form ofstereolithography (.stl) files to create potential array points whichare the basis of the simulation environment in SIMION. Because .stl isthe native file format accepted by most software packages to preparefiles for 3D printing, the same files may be used for both electrodeproduction and trajectory simulation.

Example 8: System Validation

The ability to generate, transmit, focus, and detect ions in air withthe curved polymeric electrode system was demonstrated by spraying amixture of tetralkylammonium (TAA) bromide salts (10 μM in acetonitrile)from a nanoESI emitter into the source region (E_(source)) of the curvedelectrode. Plots of ion intensity at the exit region of the curvedelectrode system were reconstructed from data obtained by scanning anIONCCD (atmospheric pressure ion detector, commercially available fromOI Analytical) detector across the exit orifice of the final electrodeat a fixed rate (FIG. 6). Reconstructed intensity plots are shown inFIG. 7 panels A-D along with the simulated ion intensities under thesame conditions.

Differences in the reconstructed intensity vs. the simulated intensitycan be largely attributed to scanning artefacts due to the width of theIONCCD (atmospheric pressure ion detector, commercially available fromOI Analytical) pixels and the stainless steel housing of the IONCCD(atmospheric pressure ion detector, commercially available from OIAnalytical). Moreover, the true distribution of ions near the emittertip is unknown and for simplification purposes ions were initiated with3D Gaussian distribution (σ_(xyz)=5 mm) in the source region and nosimulations took space charge into account. The data show that simulatedand experimental ion intensities are in good agreement, which highlightsthe utility of the SIMION-SDS algorithm in predicting the performance ofthe 3D printed polymeric electrodes.

Example 9: Separation of Ions in Air

The separation of gas-phase ions is most commonly done based on theirmass-to-charge ratio (m/z), in the case of MS, or on the basis of theirinteraction with a background gas in combination with electric fields asis done in IMS experiments. A vacuum is necessary in order to accomplishseparation based on m/z, while a laminar flowing gas is used in theinstance of an ion mobility separation.

Systems of the invention are able to perform separation of ions in airwithout the use of a vacuum or a flowing gas. Instead, pulsed voltageswere employed with the electrode system as a means to inject ions intothe curved ion path and effect a separation of tetraalkylammonium (TAA)cations. A solution of 10 μM each of tetrapropyl-, tetrabutyl-,tetrahexyl-, and tetradodecylammonium bromide in ACN was sprayed with ananoESI emitter into E_(source) The electrode assembly was modified toinclude an injection region immediately after E_(source) consisting of 2stainless steel woven meshes separated by 3 mm (FIG. 4 panel B). Afloated high voltage pulse (2530 V high, 2480 V low) was applied to themesh directly after the source region with the second mesh held flush tothe opening of the first curved electrode to facilitate electricalcontact with E₁ (FIG. 4 panel B). A pulse width of 50 ms with arepetition rate of 1 Hz was used for ion injection. Potentials appliedto the nanoESI electrode, E_(source), E₁, E₂, and E₃ were 4.50 kV, 3.20kV, 2.50 kV, 2.33 kV, and 1.45 kV, respectively.

A simulation of the separation was performed under identical conditionswith all ions originating in the space between the woven meshes.Consecutive scans of the ion trap (FIG. 8) at a 10 Hz scan frequencyshow separation of the TAA cations which agree well with the simulateddata (FIG. 9). This data shows the ability to separate gas-phase ions inair and highlights a use of the 3D printed electrodes.

FIG. 10 panel A is additional data showing separation of tetraalkylammonium cations (C₃, C₄, C₆, C₁₂) by applying a square wave, floatedhigh voltage pulse (2950 V high, 2935 V low) to the source electrode.Spectra in FIG. 10 panels B and C are subsequent scans from the trap ata 7.7 Hz acquisition rate.

Example 10: Ion/Molecule Reactions

Ion/molecule reactions (IMR) have been shown to have useful analyticalcharacteristics, especially in the case of structural elucidation. IMRsin the gas phase offer several benefits compared to their solutioncounterparts. Very little neutral reagent is required for an IMR andoften the headspace vapour is sufficient to generate measurable product.Reaction rates and efficiencies are also inherently high for most IMRs,meaning that analytes in trace quantities will still form a detectableproduct. This is especially true for IMRs performed at atmosphericpressure in IMS instruments, as the number of collisions per second isdramatically increased in comparison to the same reaction performed inan ion trap under vacuum. However, the lack of straightforwardidentification of products in IMS generally requires the use of tandemIMS-MS instrumentation. Often, significant modification to MSinstruments must be made in order to perform ion/molecule reactions,which can be costly and time-consuming. The coupling of IMS to MSinstruments suffers from similar drawbacks.

The data herein demonstrate an ion-molecule reaction performed with theplastic electrodes in air, protonated tert-butylamine andcyclohexylamine ions were generated by nanoESI from 10 ppm solutions inmethanol and reacted with dimethyl methylphosphonate (DMMP) in the lastregion (E₃) of the electrode system shown in FIG. 1A. DMMP vapor wasintroduced by replacing the final electrode with an electrode containinga hole on the far side of the swept radius of curvature (FIG. 4 panel A)and inserting a cotton swap saturated with a solution of 1000 μg/mL DMMPin methanol (FIG. 1A). The electrodes were positioned with a 3 axismoving stage such that the exiting ions were sampled with the API of anLTQ linear ion trap (Thermo).

When the ion beam exiting the electrode structure was precisely alignedwith the inlet of the MS it was found that the signal recorded by themass spectrometer was largely independent of the position of the nanoESIspray tip within E_(source) as the mass spectra and recorded intensityremained stable while adjusting the spray tip location. Mass spectra ofthe products of these two reactions sampled from the last electrode ofthe polymeric electrode assembly are shown in FIG. 11. Similar IMRsusing analogs of DMMP have previously been demonstrated for theidentification of amino functionalities in a Fourier transform ioncyclotron resonance (FT-ICR) mass spectrometer. These reactionshighlight the potential usefulness of ion manipulation outside the massspectrometer in exploring ion/molecule reactions for functional groupidentification. The ability to perform and interrogate these reactionsoutside the MS may allow for a condition in which an ion separation isperformed at atmospheric pressure after a reaction has taken place toidentify the presence of a target compound in the analyte mixture.

Another ion/molecule reaction was performed using vapor of 3-octyne asthe neutral molecule supplied by evaporation from a swab placed near the3rd turning electrode. The reacting ions were Au[CH₃CN]²⁺ generated byelectrolytic spray ionization from a gold electrode in acetonitrile. Theproduct ions were the bis-ligated 3-octyne and it quantitativelyreplaced the bis-acetonitrile FIG. 12 panels A-B). FIG. 12 panels A-Bare spectra of the ion-molecule reaction in air using electrolyticnanoESI (Au electrode) to generate Au(ACN)²⁺ from acetonitrile (ACN)spray solvent Neutral: 3-octyne on cotton swab (Ion detection: ThermoLTQ). Referring still to FIG. 12 panels A-B, the ion-molecule reactionis shown above the spectra. Note in FIG. 12 panels A-B the presence ofunassigned ions in the reagent ion spectrum. These do not react so occurunchanged in the product spectrum.

Example 11: Focusing of Ions in Air Using a Cylindrical Electrode with aCoaxial Disc Electrode

FIG. 13 illustrates simulated ion trajectories of protonatedacetonitrile ions in a cylinder of 30 mm inner diameter thatincorporates a coaxial disc electrode of 15 mm diameter and 1.6 mm thickpositioned flush to the end of the cylinder. Both the disc and cylinderare held at the same potential. When these coaxial electrodes arepositioned in close proximity to a surface held at a significantly lowerpotential (100V down to several kV lower), electric fields are createdthat cause ions to be focused to an annulus with a diameter that islargely dictated by the diameters of the central disc and outercylinder. From here on, this configuration of electrodes and applicationof appropriate potentials will be referred to as a radial ion lens.Simulated and experimental data also show that the potential offset(between electrode components and deposition/detection surface) alsoaffects both the line width of the annulus, as well as the diameter.

This radial ion lens and the type of focused ring of ions it producesare of particular interest in cases where spray ionization may be usedto modify a surface, deposit material onto a surface, and introduce ionsinto a mass spectrometer or an ion mobility spectrometer as iteliminates line-of-sight from the spray emitter to the surface,detector, or other such target as they are physically blocked by theinner disc. This prevents the contamination of the surface, detector, ortarget with neutral species or large droplets. Additionally, this mayserve as a jet-disruptor (perturbs high velocity gas streams to limitinterference with static gas within the subsequent ion optics) in caseswhere a nebulizing, or carrier gas is used in the generation of ions ortheir subsequent transfer to a surface, target, or detector.

FIG. 14 panel A shows the radial intensity of ions at a groundeddeposition surface positioned approximately 1 mm from the opening of theradial ion lens as was determined experimentally from reconstructed twodimensional ion images obtained by scanning a pixelated charge detectoracross the opening of lens as was described previously. The tracesobtained in FIG. 14 panel A were obtained by fitting a circle to a twodimensional plot of ion intensity (such as that shown in FIG. 14 panelC, centering the data using the center of the fit circle, and convertingion intensity into polar coordinates. A histogram plot of the radial ionintensity was then made under various experimental conditions. The firstnumber in the legend displayed in FIG. 14 panel A represents thepotential difference between the radial ion lens and the detectionsurface with the second number representing the potential applied to thenanoelectrospray ion source in relation to the first number (i.e. 0.25kV/1 kV is the case in which 250 V was applied to the radial ion lensand a potential of 1.25 kV was applied to the nanoelectrospray emitter).FIG. 14 panel B shows a simplified version of the experimental setup aswell as a top down view of the radial ion lens. Small filaments are usedto connect the central disc to the walls of the cylinder.

FIG. 15 is a graph showing efficiency of ion transfer from ananoelectrospray ionization source to a grounded deposition surface bythe radial ion lens with different applied potentials. The spray currentfor each data point shown in FIG. 15 was calculated by measuring apotential drop across a 4.7 MΩ resistor which by Ohm's law may becalculated using the equation shown below:

${{Current}(A)} = \frac{{Potential}{drop}{across}{resistor}(V)}{4.7 \times 10^{6}\Omega}$

The corresponding deposition current was measured using a picoammeterconnected to a copper sheet used as the deposition surface. Theefficiency was then taken by dividing the deposition current by thecalculated spray current under a given condition. Different spraycurrents were achieved by adjusting the potential applied to ananoelectrospray emitter filled with a 2 mM equimolar mixture oftetrabutyl-, tetrahexyl-, tertraoctyl-, and tetradodecylammonium halidesin a solution of 4:1 methanol:acetonitrile with 0.1% formic acid. Thespray emitter was positioned as shown in FIG. 14 panel B.

Example 12: 3D Printed Ion Mobility Spectrometer

A 3D printed ion mobility spectrometer (IMS) was constructed by fuseddeposition modeling. A glycol-modified polyethylene terephthalate (PETg)impregnated with carbon nanotubes was used for electrode components(shown as black in FIG. 16) and polylactic acid (PLA) was used toconstruct electrode housing components (shown as hatched in FIG. 16).The IMS consisted of 4 parts:

-   -   (a) A source/focal region that contained the nanoelectrospray        ion source as well as the radial ion lens. This portion is shown        as (a) in FIG. 16.    -   (b) An ion injection region consisting of an electrode with a        stainless steel mesh held flush to the electrode on the side        immediately preceding the drift cell and separated from the        first drift electrode by a PLA spacer. Ion injection was        achieved by applying a floated high voltage pulse the injection        electrode. During the high portion of this pulse (typically        50-100 V in relation to the potential on the first drift ring)        ions are extracted from the radial ion lens and injected into        the drift cell. During the low portion of the pulse (typically        −25 V in relation to the potential applied on the first drift        electrode) ions are blocked from entering the drift cell. This        portion is shown as (b) in FIG. 16.    -   (c) A drift cell consisting of 30 electrodes and separated by        spacers incorporated in the housing assembly. A potential        gradient is applied along the drift cell so as to create a        uniform electric field which drives ions toward the detector        region. The entrance and exit of the drift cell each incorporate        a stainless steel mesh held flush with the surface. The mesh on        the first drift electrode ensures the changing potential on the        injection electrode does not interfere with the electric field        within the drift region. The mesh on the final drift electrode        serves to shield the detector surface from the approaching ion        packets, greatly reducing effects due to an image charge on the        detector surface. The exit electrode and mesh are generally held        at a potential of 100-500 V relative to the grounded detection        surface. This portion is shown as (c) in FIG. 16.    -   (d) The detection region consists of a Faraday plate constructed        of a suitable conductor (in this case a copper sheet) which is        connected to a current amplifier, read by an oscilloscope. The        detector region is not limited to detection by this manner and        may incorporate any manner of suitable charge detector capable        of operating at atmospheric pressure or above.        Electrodes used for injection and drift cell construction were        similar in design to the radial ion lens previously described;        however, the height of the outer cylindrical portion was matched        to that of the central disc.

FIG. 17 panel A shows both a mass spectrum of ions created byelectrospraying a 2 mM equimolar solution of tetrabutyl-, tetrahexyl-,tertraoctyl-, and tetradodecylammonium halides in 4:1methanol:acetonitrile with 0.1% formic acid after they are focused intoan annulus by the radial ion lens, continuously injected (constantV_(inject)) into a drift cell, and subsequently exit the drift regionwhere they are sampled by a mass spectrometer. A separation of ions fromthis same mixture was accomplished by injecting ions into the drift celland recording their arrival times at the detection surface. FIG. 17panel B shows the results of this separation when injection durationsranging from 0.3 to 1.0 ms were used.

1-21. (canceled)
 22. A system for manipulating a sample, the systemcomprising: an ion generator configured to generate ions from a sample;an ion separator configured to separate at or above atmospheric pressurethe ions received from the ion generator, wherein the ion separator iscomprised of a plurality of triangular electrode segments that operablyalign with each other to create a curved path for the generated ions;and an ion focusing element that focuses the separated ions from the ionseparator at or above atmospheric pressure.
 23. The system according toclaim 22, further comprising a substrate positioned to the receive thefocused separated ions at one or more discrete locations on thesubstrate.
 24. The system according to claim 22, further comprising adetector that receives and detects the focused separated ions.
 25. Thesystem according to claim 24, wherein the ion generator comprises: anionization source; and an ion injector configured to interface with theionization source such that ions produced by the ionization source arereceived by the ion injector.
 26. The system according to claim 25,wherein the ionization source is out-of-line with the detector.
 27. Thesystem according to claim 25, wherein the ion injector is maintained ator above atmospheric pressure.
 28. The system according to claim 25,wherein the ion injector comprises a cavity and one or more wire meshesthat receive the ions produced by the ionization source.
 29. The systemaccording to claim 25, wherein the ion injector receive the ionsproduced by the ionization source and transmits them as a focused beamto the ion separator.
 30. The system according to claim 30, wherein theion separator comprises three triangular electrode segments.
 31. Thesystem according to claim 30, wherein the ion separator furthercomprises an insulating material between each of the three triangularelectrode segments
 32. A method for manipulating sample ions, the methodcomprising: generating ions from a sample at or above atmosphericpressure; separating the ions at or above atmospheric pressure using anion separator configured to separate at or above atmospheric pressurethe ions received from the ion generator, wherein the ion separator iscomprised of a plurality of triangular electrode segments that operablyalign with each other to create a curved path for the generated ions;and focusing the separated ions at or above atmospheric pressure,thereby manipulating the sample ions.
 33. The method according to claim32, further comprising depositing the focused separated ions at one ormore discrete locations on a substrate.
 34. The method according toclaim 32, further comprising detecting the focused separated ions. 35.The method according to claim 34, wherein the detecting step is at orabove atmospheric pressure.
 36. The method according to claim 34,wherein the detecting step is below atmospheric pressure.
 37. The methodaccording to claim 34, wherein detecting comprises receiving the ionsinto a mass spectrometer or a miniature mass spectrometer.
 38. Themethod according to claim 32, wherein the ion separator comprises threetriangular electrode segments.